Abstract P2110: Myosin-7 E848G Mutation Reduces Contractility And Activates P53-associated Intrinsic Apoptosis In Patient-derived Induced Pluripotent Stem Cell Model Of Hypertrophic Cardiomyopathy

Abstract

Introduction: Familial hypertrophic cardiomyopathy (HCM), affecting 1 in 500 adults, is characterized by idiopathic thickening of the heart and impaired systolic function. Mechanisms through which cardiac sarcomeric mutations manifest in HCM are poorly understood. Hypothesis: We previously identified a novel MYH7 E848G mutation associated with HCM that manifested as left ventricular hypertrophy, diffuse fibrosis, and severe systolic dysfunction. We hypothesize that the E848G variant impairs tissue contractility and causes cell death in a dose-dependent manner. Methods: We created MYH7-EGFP expressing cardiomyocytes (CMs) with MYH7 wt/wt-EGFP , MYH7 wt/E848G-EGFP , or MYH7 E848G/E848G-EGFP alleles using patient-derived induced pluripotent stem cells (hiPSCs), CRISPR-Cas9 gene-editing, and standard differentiation protocols. CMs were cocultured with stromal cells to create 3D engineered heart tissues (EHTs) or cultured as a monolayer in high calcium stress media. EHTs were enzymatically digested and GFP+ cardios were sorted for analysis. Results: Monolayer MYH7 wt/E848G-EGFP CMs had higher TUNEL + percentage (13.1 ± 2.1%) at d33 and lower cell survival (52.0 ± 6.6% of d30 seeded) at d42 relative to MYH7 wt/wt-EGFP (6.6 ± 2.6%; 107 ± 2.1%). Screening revealed elevated levels of proteins associated with intrinsic apoptosis (p53, 1.93 ± .03-fold; p21, 1.66 ± .10-fold; Bax, 1.56 ± .11-fold) in MYH7 wt/E848G-EGFP CMs at d35 relative to MYH7 wt/wt-EGFP . MYH7 wt/E848G-EGFP (n = 20; 67.5 ± 9.0 μN) had reduced maximum active twitch force relative to MYH7 wt/wt-EGFP (n = 19; 226 ± 17 μN) 14 days post-cast. MYH7 wt/E848G-EGFP EHTs had lower percentage of EGFP + cells (13.5 ± 0.8%) 7 days post-cast relative to MYH7 wt/wt-EGFP (25.8 ± 0.8%). EGFP + cells sorted from 7 day post-cast MYH7 wt/E848G-EGFP EHTs had elevated TP53 transcription (1.67 ± .29-fold) relative to MYH7 wt/wt-EGFP . Conclusion: These results suggest MYH7 E848G mutation reduces sarcomere contractility and induces p53-associated cell death. High calcium stress media induces cell death that likely contributes to contractile dysfunction seen in MYH7 E848G HCM. Ongoing studies will elucidate mechanisms through which E848G activates intrinsic apoptosis.